Green processing technique for sea cucumber powder capable of achieving directional enrichment and improving polysaccharide release rate

Abstract

The present invention belongs to the field of food processing, and discloses a green processing technique for a sea cucumber powder capable of achieving directional enrichment and improving the polysaccharide release rate. The sea cucumber powder is prepared by the following steps: S1, pretreating sea cucumbers; S2, enriching nutrient substances in the sea cucumbers; S3, regulating and controlling the particle size of a sea cucumber powder; S4, performing a "single-stage" supercritical treatment; S5, performing in situ ultramicro physical crushing; and S6, performing a "dual-stage" supercritical treatment. In the present invention, by means of the design of a gas distributor and the additional arrangement of a second perforated plate, a supercritical extractor can be used to perform effective supercritical carbon dioxide extraction on an ultra-micro sea cucumber powder, thereby achieving the deodorization of the ultra-micro sea cucumber powder and improving the edible quality thereof. Moreover, by detecting and regulating the bulk density and the tap density, optimal processing parameters for the ultra-micro sea cucumber powder are found, such that the prepared ultra-micro sea cucumber powder has a high polysaccharide release rate and good edible quality, and can be easily processed subsequently.

Description

A green processing technology for sea cucumber powder with targeted enrichment to enhance polysaccharide release rate. Technical Field

[0001] This invention relates to a green processing technology for sea cucumber powder that uses targeted enrichment to improve polysaccharide release rate, and belongs to the field of food processing. Background Technology

[0002] Sea cucumber (Stichopus japonicus) belongs to the class Holothuroidea, phylum Mollusca, and is a benthic marine organism, a valuable marine biological resource. Sea cucumbers are known for their high protein and low fat content, and also contain abundant trace elements and various amino acids. Sea cucumber polysaccharides are one of the important active components of the sea cucumber's body wall, accounting for 4% to 10% of the total organic matter in dried sea cucumbers. Sea cucumber polysaccharides are acidic mucopolysaccharides with strong biological activities in anticoagulation, lowering blood lipids, regulating immunity, and anti-aging. However, sea cucumber polysaccharides are bound to proteins through glycopeptide bonds and hydrogen bonds; therefore, the absorption and utilization of polysaccharides require the degradation of the proteins they are linked to.

[0003] In existing technologies, to achieve effective separation of the two, the concept of ultrafine sea cucumber powder has been proposed. For example, CN 106509694 A – Preparation of a convenient and easily digestible ultrafine sea cucumber powder – discloses improved digestibility and a high retention rate of crude polysaccharides (95.42%). CN 105495411 A – Preparation method of ultrafine pure sea cucumber powder tablets – discloses that the 1-30μm ultrafine sea cucumber powder achieves cell disruption, allowing for the complete release of active substances. However, the processing technology of the ultrafine sea cucumber powder in the aforementioned two patents is relatively rudimentary. It involves simple washing, sterilization, and enzyme inactivation followed by direct ultrafine pulverization, lacking a deodorization step. The product still retains a large number of fishy-smelling molecules, and there is no indicator to measure the particle size of the pulverized sea cucumber powder. Therefore, the final product has questionable edibility, poor purity, and difficult quality control.

[0004] Other methods for separating proteins and polysaccharides include protease hydrolysis and alkaline extraction, such as the method disclosed in patent CN 110204626 A – an apparatus and method for separating and purifying sea cucumber polysaccharides. However, using alkaline solutions can damage the structure of sea cucumber polysaccharides, causing the loss of glycosyl groups and altering the polysaccharide structure. Therefore, it is necessary to strictly control the concentration of the alkaline solution and the reaction conditions. Enzymatic hydrolysis requires control of the enzyme concentration, hydrolysis temperature, and time. This method is cumbersome to operate, and the enzymatic hydrolysis products have unpleasant flavors, affecting the taste. Therefore, physical pulverization is preferable to enzymatic hydrolysis.

[0005] Therefore, the extraction and utilization of sea cucumber polysaccharides still need further research in order to produce a sea cucumber polysaccharide product with good edible quality and excellent product quality, and to use a more superior and effective method. Summary of the Invention Technical issues

[0006] Sea cucumber polysaccharides have high utilization value, but to utilize them, it is necessary to separate them from proteins. Common separation methods include physical pulverization and enzymatic extraction. Physical pulverization is simpler than enzymatic extraction, and product quality is easier to control. However, current physical pulverization techniques are still relatively rudimentary, resulting in sea cucumber powder of questionable quality, poor purity, and difficulty in quality control. Therefore, improvements are needed.

[0007] Technical content

[0008] The purpose of this invention is to overcome the shortcomings of existing sea cucumber polysaccharide and protein separation technologies and provide a method for preparing sea cucumber powder with a high polysaccharide release rate. This invention improves the sea cucumber polysaccharide release rate by employing a suitable physical pulverization technique, preparing a sea cucumber powder with a high polysaccharide release rate and anti-atherosclerotic properties, which is verified using simulated digestion and animal models.

[0009] To achieve the above objectives, the present invention provides a method for preparing sea cucumber powder with a high polysaccharide release rate, wherein the sea cucumber powder is prepared through the following steps:

[0010] S1. Sea cucumber pretreatment: Make an opening in the abdomen of the sea cucumber, remove the contents of the sea cucumber body cavity, and wash the sea cucumber body wall with water to obtain pretreated sea cucumber;

[0011] S2. Enrichment of nutrients in sea cucumbers: The pretreated sea cucumbers obtained in step S1 are temporarily kept in warm water and then dried to obtain dried sea cucumber products.

[0012] S3. Particle size control of sea cucumber powder: The dried sea cucumber obtained in step S2 is pulverized into coarse sea cucumber powder of 5-30 mesh.

[0013] S4, “Single” supercritical: The sea cucumber coarse powder obtained in step S3 is placed in the extraction vessel of a supercritical extractor for supercritical carbon dioxide extraction to remove the fishy smell.

[0014] S5. In-situ ultrafine physical pulverization: The sea cucumber powder that has been deodorized in step S4 is pulverized into ultrafine powder to obtain powder. The bulk density of the powder is controlled to be 0.65-0.70 g / mL, and the tap density is 0.70-0.80 g / mL.

[0015] S6, "Double" supercritical: The sea cucumber powder obtained in step S5 is placed in the extraction vessel of a supercritical extractor and subjected to supercritical carbon dioxide extraction to remove the fishy smell, resulting in sea cucumber powder with a high polysaccharide release rate.

[0016] Furthermore, the temperature of the warm water mentioned in step S2 is 50-60°C.

[0017] Furthermore, the temporary incubation time described in step S2 is 4 to 8 hours.

[0018] Furthermore, the warm water temporary holding treatment in step S2 is as follows: the pretreated sea cucumbers are placed in warm water at 50-60℃ for 4-8 hours, and the warm water is changed every 0.5-1 hour.

[0019] Furthermore, the drying process in step S2 includes freeze drying, heat drying, or air drying.

[0020] Furthermore, the supercritical extractor mentioned in steps S4 and S6 is a supercritical extractor equipped with a gas distributor.

[0021] Furthermore, the gas distributor in the supercritical fluid extractor is an improved gas distributor.

[0022] Furthermore, the improved gas distributor includes an installation pipe (1), a connecting flange (2), a first perforated plate (3), a filter screen (4), a support column (5), a baffle (6), a telescopic rod (7), a spring (70), a pressure plate (8), and a second perforated plate (9).

[0023] The installation tube (1) is made of transparent material, making it easy to observe the internal condition. Both the upper and lower outer sides of the installation tube (1) are connected to connecting flanges (2) for easy connection to external pipes. The inner middle and lower inner sides of the installation tube (1) are connected to first perforated plates (3). A filter screen (4) is connected to the lower side of each first perforated plate (3). Four support columns (5) are connected between the first perforated plates (3). A baffle (6) is connected to the upper side of each support column (5). The baffles (6) are all connected to the installation tube (1). The first hole plate (3) and the second hole plate (9) are connected to each other. The first hole plate (3) and the second hole plate (9) are the same shape. The second hole plate (9) has twelve conical parts on its upper side to facilitate reflux.

[0024] Furthermore, in step S4, the parameters of the extraction vessel are set as follows: 30-40℃, extraction pressure: 25-30MPa, CO2 flow rate: 25-30L / h, and extraction time: 2-2.5h.

[0025] Furthermore, in step S5, the material is pre-cooled before being pulverized using an ultrafine pulverizer; the pre-cooling is performed using dry ice, liquid nitrogen, or by placing the material in a freezing environment; the temperature of the freezing environment is -80 to -20 degrees Celsius.

[0026] Furthermore, in step S5, the ultrafine powder is pulverized at a speed of 8000-12000 rpm using a sieve with a mesh size of 0.3-0.6 mm.

[0027] Furthermore, in step S5, the bulk density is 0.67–0.70 g / mL and the tap density is 0.77–0.80 g / mL.

[0028] Specifically, in step S5, the bulk density is 0.70 g / mL and the tap density is 0.80 g / mL.

[0029] Furthermore, in step S6, the parameters of the extraction vessel are set as follows: 30-40°C, extraction pressure: 25-30 MPa, CO2 flow rate: 25-30 L / h, and extraction time: 2-2.5 h.

[0030] This invention provides sea cucumber powder with a high polysaccharide release rate prepared according to the above method.

[0031] The application of the sea cucumber powder with high polysaccharide release rate provided by this invention in the fields of food, health products or pharmaceutical preparation.

[0032] Furthermore, the application in the field of health supplements is the preparation of health supplements that help maintain healthy levels of blood lipids (cholesterol / triglycerides).

[0033] Furthermore, the application in the field of drug preparation is the preparation of drugs for the prevention and treatment of atherosclerotic diseases.

[0034] The beneficial effects of this invention are:

[0035] 1. Current physical pulverization methods for protein separation from sea cucumber polysaccharides are relatively rudimentary and lack a deodorization step. This invention, through the design of a gas diffuser and the addition of a second perforated plate, enables the supercritical fluid extractor to effectively extract ultrafine sea cucumber powder using supercritical carbon dioxide, thereby deodorizing the powder and improving its edible quality. Furthermore, the second perforated plate design allows for the reflux of carbon dioxide fluid for secondary extraction, further improving extraction efficiency and effectively removing fishy-smelling molecules from the ultrafine sea cucumber powder, thus increasing the purity of the sea cucumber polysaccharides in the product.

[0036] 2. Currently, the processing technology for ultrafine sea cucumber powder lacks parameter measurement and quality control. This invention, through the detection and control of bulk density and tapped density, has discovered the optimal processing parameters for ultrafine sea cucumber powder, resulting in a powder with high polysaccharide release rate, good edible quality, and ease of subsequent processing. Furthermore, the obtained ultrafine sea cucumber powder also exhibits excellent anti-atherosclerotic and lipid-lowering effects, making it a product with broad market application prospects. Attached Figure Description

[0037] Figure 1 is a three-dimensional structural diagram of the improved gas distributor; where 1 is the mounting pipe, 2 is the connecting flange, 5 is the support column, 7 is the telescopic rod, 8 is the pressure plate, and 9 is the second orifice plate.

[0038] Figure 2 is a partial cross-sectional three-dimensional structural schematic diagram of the improved gas distributor; wherein, 1, mounting pipe, 2, connecting flange, 3, first orifice plate, 4, filter screen, 5, support column, 6, baffle, 7, telescopic rod, 8, pressure plate, 9, second orifice plate.

[0039] Figure 3 is an enlarged three-dimensional structural diagram of point A of the improved gas distributor; where 1 is the mounting tube, 5 is the support column, 6 is the baffle, 7 is the telescopic rod, 70 is the spring, 8 is the pressure plate, and 9 is the second orifice plate.

[0040] Figure 4 shows a supercritical extraction apparatus with a gas diffuser installed, where the arrow indicates the installation location of the improved gas diffuser.

[0041] Figure 5 shows the polysaccharide release curves of sea cucumbers in Examples 1, 2 and Comparative Example 1;

[0042] Figure 6 shows the cholesterol levels in the blood of mice after intervention with high-fat diet in Example 1 and Comparative Example 1 (different letters indicate significance);

[0043] Figure 7 shows aortic slices of mice treated with high-fat diet in Example 1 and Comparative Example 1 after intervention.

[0044] Figure 8 shows the blood vessel wall thickness of mice after intervention with high-fat diet in Example 1 and Comparative Example 1 (different letters indicate significance);

[0045] Figure 9 is a two-dimensional differential topographic map of GC-IMS in Example 1;

[0046] Figure 10 is a comparative 3GC-IMS two-dimensional differential topographic map;

[0047] Figure 11 is a comparative 4GC-IMS two-dimensional differential topographic map. Detailed Implementation

[0048] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] In this invention, unless otherwise explicitly specified and limited, the terms "connected," "linked," and "fixed" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0050] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on top" of the second feature includes the first feature directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature includes the first feature directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.

[0051] Experimental instruments

[0052] The supercritical fluid extractor equipped with an improved gas diffuser used in the examples and comparative examples was manufactured by Jiangsu Gaoke Pharmaceutical Equipment Co., Ltd., and its model number is GKSCFE220-40-30L. The gas diffuser of this supercritical fluid extractor was improved, and the improved gas diffuser is shown in Figures 1-3.

[0053] The improved gas distributor includes an installation pipe 1, a connecting flange 2, a first orifice plate 3, a filter screen 4, a support column 5, a baffle 6, a telescopic rod 7, a spring 70, a pressure plate 8, and a second orifice plate 9.

[0054] The installation tube 1 is made of transparent material, making it easy to observe the internal situation. Both the upper and lower outer sides of the installation tube 1 are connected to connecting flanges 2, which facilitates the connection of external pipes. The inner middle and lower inner sides of the installation tube 1 are connected to first perforated plates 3. The lower side of each first perforated plate 3 is connected to a filter screen 4. Four support columns 5 are connected between the first perforated plates 3. Each support column 5 is connected to a baffle 6, which is in contact with the installation tube 1. Each baffle 6 is connected to a telescopic rod 7. A spring 70 is connected between the fixed end and the telescopic end of the telescopic rod 7. Each telescopic end of the telescopic rod 7 is rotatably connected to a pressure plate 8. A second perforated plate 9 is placed between the baffles 6. The pressure plate 8 is in contact with the second perforated plate 9. The first perforated plate 3 and the second perforated plate 9 have the same shape. The upper side of the second perforated plate 9 is provided with twelve conical parts to facilitate backflow.

[0055] When performing supercritical carbon dioxide extraction, an improved gas distributor is used to move the device to the area where the extracted components need to be extracted, connecting the lower flange 2 of the mounting tube 1 to the feed pipe. The mounting tube 1 is made of transparent material for easy observation of the internal structure. Then, the pressure plate 8 is pulled and rotated, causing the telescopic rod 7 to extend and the spring 70 to be stretched. Next, the second perforated plate 9 contacts the baffle 6. Then, the pressure plate 8 is rotated in the opposite direction to reset, and then the pressure plate 8 is released. The spring 70 rebounds, and the telescopic rod 7 retracts, bringing the pressure plate 8 into contact with the second perforated plate 9. This secures the second perforated plate 9, preventing it from falling off when the carbon dioxide fluid flows. Finally, the upper connecting flange 2 connects to the feed inlet of the extraction tank. By adding carbon dioxide fluid into the feed pipe, the carbon dioxide fluid enters the installation pipe 1, flows through the first perforated plate 3, the filter screen 4, and the second perforated plate 9, and then flows into the extraction tank to extract the material. The first perforated plate 3 and the second perforated plate 9 have the same shape, which can uniformly feed the carbon dioxide fluid, improve the extraction efficiency and production efficiency. At the same time, the device can be placed in the extraction tank, and the carbon dioxide can achieve a backflow effect under the action of the conical part. Thus, under the action of the conical part on the second perforated plate 9, the carbon dioxide fluid backflows for secondary extraction, which facilitates secondary extraction of the material. It also reduces the pressure when the carbon dioxide fluid is discharged from the material basket, prevents the material from being carried out, and improves the extraction effect.

[0056] Example 1

[0057] S1. Sea cucumber pretreatment: Make an opening in the abdomen of the fresh sea cucumber, remove the contents of the sea cucumber body cavity, and wash the sea cucumber body wall with water;

[0058] S2. Enrichment of nutrients in sea cucumbers: The pretreated sea cucumbers obtained in step S1 are temporarily kept in warm water at 60℃ for 6 hours, and the water is changed every 0.5 hours to remove salt from the sea cucumbers. Then the sea cucumbers are taken out and dried in a vacuum freeze dryer for 24 hours to remove moisture and obtain dried sea cucumber products.

[0059] S3. Particle size control of sea cucumber powder: The dried sea cucumber is pulverized into 20-mesh coarse sea cucumber powder in a pulverizer, ball mill or crusher;

[0060] S4, “Single” Supercritical: Deodorize the sea cucumber powder using a supercritical extractor equipped with an improved gas diffuser. Place the sea cucumber powder from step S3 into the extraction vessel of the supercritical extractor. Set the extraction vessel temperature to 30℃, the extraction pressure to 30MPa, the CO2 flow rate to 30L / h, and the extraction time to 2.5h. After extraction, collect the sea cucumber powder from the extraction vessel and store it for later use.

[0061] S5. In-situ ultrafine physical pulverization: The sea cucumber powder obtained in step S4 was pre-cooled in liquid nitrogen for 1 hour, and then pulverized using an ultrafine pulverizer. The ultrafine pulverizer was pulverized at a speed of 12000 rpm and a 0.6 mm mesh screen. The bulk density of the powder was controlled to be 0.65 g / mL and the tap density was 0.73 g / mL.

[0062] S6, "Double" Supercritical: Deodorize the sea cucumber powder using a supercritical extractor equipped with an improved gas diffuser. Place the sea cucumber powder from step S5 into the extraction vessel of the supercritical extractor. Set the extraction vessel temperature to 30℃, the extraction pressure to 30MPa, the CO2 flow rate to 30L / h, and the extraction time to 2.5h. After extraction, collect the sea cucumber powder from the extraction vessel and store it for later use.

[0063] S7. Package, sterilize, and seal the sea cucumber powder obtained in step S6, and cool it to room temperature to obtain the ultrafine sea cucumber powder product.

[0064] Example 2

[0065] S1. Sea cucumber pretreatment: Make an opening in the abdomen of the harvested sea cucumber, remove the contents of the sea cucumber body cavity, and wash the sea cucumber body wall with water.

[0066] S2. Enrichment of nutrients in sea cucumbers: The pretreated sea cucumbers obtained in step S1 are temporarily kept in warm water. The sea cucumbers are kept in warm water at 60℃ for 6 hours, and the water is changed every 0.5 hours to remove salt from the sea cucumbers. Then the sea cucumbers are taken out and dried in a vacuum freeze dryer for 24 hours to remove moisture and obtain dried sea cucumber products.

[0067] S3. Particle size control of sea cucumber powder: The dried sea cucumber is pulverized into 20-mesh coarse sea cucumber powder in a pulverizer, ball mill or crusher;

[0068] S4, “Single” Supercritical: Deodorize the sea cucumber powder using a supercritical extractor equipped with an improved gas diffuser. Place the sea cucumber powder from step S3 into the extraction vessel of the supercritical extractor. Set the extraction vessel temperature to 30℃, the extraction pressure to 30MPa, the CO2 flow rate to 30L / h, and the extraction time to 2.5h. After extraction, collect the sea cucumber powder from the extraction vessel and store it for later use.

[0069] S5. In-situ ultrafine physical pulverization: The sea cucumber powder obtained in step S4 was pre-cooled in liquid nitrogen for 1 hour, and then pulverized using an ultrafine pulverizer. The ultrafine pulverizer was pulverized at a speed of 12000 rpm and a 0.6 mm mesh screen. The bulk density of the powder was controlled to be 0.70 g / mL and the tap density was 0.80 g / mL.

[0070] S6, "Double" Supercritical: Deodorize the sea cucumber powder using a supercritical extractor equipped with an improved gas diffuser. Place the sea cucumber powder from step S5 into the extraction vessel of the supercritical extractor. Set the extraction vessel temperature to 30℃, the extraction pressure to 30MPa, the CO2 flow rate to 30L / h, and the extraction time to 2.5h. After extraction, collect the sea cucumber powder from the extraction vessel and store it for later use.

[0071] S7. Package, sterilize, and seal the sea cucumber powder obtained in step S6, and cool it to room temperature to obtain the ultrafine sea cucumber powder product.

[0072] Example 3

[0073] S1. Sea cucumber pretreatment: Make an opening in the abdomen of the harvested sea cucumber, remove the contents of the sea cucumber body cavity, and wash the sea cucumber body wall with water.

[0074] S2. Enrichment of nutrients in sea cucumbers: The pretreated sea cucumbers obtained in step S1 are temporarily kept in warm water. The sea cucumbers are kept in warm water at 60℃ for 6 hours, and the water is changed every 0.5 hours to remove salt from the sea cucumbers. Then the sea cucumbers are taken out and dried in a vacuum freeze dryer for 24 hours to remove moisture and obtain dried sea cucumber products.

[0075] S3. Particle size control of sea cucumber powder: The dried sea cucumber is pulverized into 20-mesh coarse sea cucumber powder in a pulverizer, ball mill or crusher;

[0076] S4, “Single” Supercritical: Deodorize the sea cucumber powder using a supercritical extractor equipped with an improved gas diffuser. Place the sea cucumber powder from step S3 into the extraction vessel of the supercritical extractor. Set the extraction vessel temperature to 30℃, the extraction pressure to 30MPa, the CO2 flow rate to 30L / h, and the extraction time to 2.5h. After extraction, collect the sea cucumber powder from the extraction vessel and store it for later use.

[0077] S5. In-situ ultrafine physical pulverization: The sea cucumber powder obtained in step S4 was pre-cooled in liquid nitrogen for 1 hour, and then pulverized using an ultrafine pulverizer. The ultrafine pulverizer was pulverized at a speed of 12000 rpm and a 0.6 mm mesh screen. The bulk density of the powder was controlled to be 0.75 g / mL and the tap density was 0.87 g / mL.

[0078] S6, "Double" Supercritical: Deodorize the sea cucumber powder using a supercritical extractor equipped with an improved gas diffuser. Place the sea cucumber powder from step S5 into the extraction vessel of the supercritical extractor. Set the extraction vessel temperature to 30℃, the extraction pressure to 30MPa, the CO2 flow rate to 30L / h, and the extraction time to 2.5h. After extraction, collect the sea cucumber powder from the extraction vessel and store it for later use.

[0079] S7. Package, sterilize, and seal the sea cucumber powder obtained in step S6, and cool it to room temperature to obtain the ultrafine sea cucumber powder product.

[0080] Example 4

[0081] S1. Weigh 50g of the ultrafine sea cucumber powder and 30g of microcrystalline cellulose prepared in Example 1, put them into a mixer, and mix for 15 minutes at a speed of 1000r / min to make the two powders evenly dispersed.

[0082] S2. Add 2g of magnesium stearate to the mixer, adjust the speed to 800r / min, and continue mixing for 5min to ensure that the magnesium stearate is evenly covered on the powder surface and to reduce the phenomenon of sticking to the wall during the filling process.

[0083] S3. Place the empty capsules in an environment of 25℃ and 45%-55% relative humidity for 2 hours to equilibrate. Then start the capsule filling machine and adjust the filling amount to 0.5g / capsule, of which the content of ultrafine sea cucumber powder is about 0.3g / capsule. The capsule filling machine speed is 30r / min and the filling pressure is 0.3MPa to obtain a capsule for helping to maintain healthy blood lipid levels or to prevent and treat atherosclerosis.

[0084] Comparative Example 1

[0085] S1. Sea cucumber pretreatment: Open the abdomen of the harvested sea cucumber, remove the contents of the sea cucumber body cavity, and wash the sea cucumber body wall with water to obtain dried sea cucumber.

[0086] S2. Enrichment of nutrients in sea cucumbers: The pretreated sea cucumbers obtained in step S1 are temporarily kept in warm water. The sea cucumbers are kept in warm water at 60℃ for 6 hours, and the water is changed every 0.5 hours to remove salt from the sea cucumbers. Then the sea cucumbers are taken out and dried in a vacuum freeze dryer for 24 hours to remove moisture.

[0087] S3. Particle size control of sea cucumber powder: The dried sea cucumber is pulverized into 20-mesh coarse sea cucumber powder in a pulverizer.

[0088] Comparative Example 2

[0089] S1. Sea cucumber pretreatment: Open the abdomen of fresh sea cucumber, remove the contents of the sea cucumber body cavity, and wash the sea cucumber body wall with water to obtain dried sea cucumber. Then take out the sea cucumber and remove the moisture by vacuum freeze drying machine for 24 hours.

[0090] S2. Particle size control of sea cucumber powder: The dried sea cucumber is pulverized into 20-mesh coarse sea cucumber powder in a pulverizer, ball mill or crusher.

[0091] Following the experimental method of Zhang Tongtong (Zhang Tongtong. Optimization of Determination Method of Sea Cucumber Polysaccharide and its In vivo Metabolism Study [D]. Dalian University of Technology, 2023.), 10 μL of sample solution was added to an ELISA plate, followed by 200 μL of DMB staining solution (accurately prepared 42.8 mg / L DMB solution, adjusted to pH 3.3 with formic acid to obtain solution A, solution A mixed with 2 mol / L Tris solution at a ratio of 10:1 to obtain DMB staining solution). The plate was shaken and incubated at room temperature in the dark for 15 min, then the absorbance was measured at 525 nm. The content of sulfated polysaccharide in the sample was calculated using a standard curve (y = 1.625x + 0.4439, R...). 2 =0.9989).

[0092] The sea cucumber powders from Examples 1, 2, and 1 (Comparative Example 1) were subjected to the above-mentioned tests. The results of the sea cucumber polysaccharide release rate are shown in Figure 5. It can be seen that the release rate of sea cucumber polysaccharides was significantly improved after ultrafine grinding. Sea cucumber polysaccharides are mainly linked to proteins, making them difficult to release into digestive juices and thus difficult for the human body to utilize. However, the physical grinding technology provided in these examples can increase the specific surface area, thereby increasing the contact area with digestive enzymes and providing more enzyme cleavage sites, thus improving the release rate of sea cucumber polysaccharides. Compared to Comparative Example 1, the polysaccharide release rate of Example 1 increased by 21%.

[0093] As the bulk density and tapped density gradually increase, the particle size of the ultrafine sea cucumber powder gradually decreases. The particle size of the ultrafine sea cucumber powder in Example 3 has reached an extremely fine level, which also means that processing to this degree is quite difficult, and the polysaccharide release rate cannot be further improved. The polysaccharide release rate of the ultrafine sea cucumber powder obtained in Example 3, as measured in the above experiments, is not significantly different from that in Example 2. In addition, microstructure experiments show that as the particle size decreases, the electrostatic force increases, and the ultrafine powder is more prone to aggregation, which will affect subsequent product processing and the edible quality of the product. Therefore, controlling the bulk density and tapped density to around 0.70 g / mL and 0.80 g / mL respectively yields the best results and should not be further increased. At this point, the polysaccharide release rate of the product is high, the processing difficulty is not high, and the edible quality is good.

[0094] Animal experiment content

[0095] 1. Laboratory animals:

[0096] 36 male APOE mice, 18-22g; 6 male C57 mice, 18-22g; Animal source: Liaoning Changsheng Biotechnology Co., Ltd., Experimental Animal Production License No.: NO.SCXK(Liaoning)2020-0001.

[0097] 2. Experimental grouping and treatment:

[0098] Experimental grouping and treatment:

[0099] (1) Normal control group: 8 C57 mice, fed normally, without gavage;

[0100] The APOE mouse strain is based on the C57 mouse. APOE mice are obtained by knocking out the APOE gene from the C57 mouse background. Generally, C57 mice are used as a blank control.

[0101] (2) Model group: 8 APOE mice were fed a high-fat diet and were not given gavage;

[0102] (3) Positive control group: 8 APOE mice were fed a high-fat diet and were given atorvastatin by gavage at a dose of 1.8 mg / kg once a day for 8 weeks at a dose of 300 uL.

[0103] (4) Comparative Example 1 High-dose group: 8 APOE mice were fed a high-fat diet and were given a high dose of ordinary sea cucumber powder by gavage, 2 mg / g, once a day for 8 weeks.

[0104] (5) Comparative Example 1 Low-dose group: 8 APOE mice were fed a high-fat diet and were given a low dose of ordinary sea cucumber powder by gavage, 1 mg / g, once a day for 8 weeks.

[0105] (6) Example 1 High-dose group: 8 APOE mice were fed a high-fat diet and were given a high dose of ultrafine sea cucumber powder by gavage, 2 mg / g, once a day for 8 weeks;

[0106] (7) Example 1 Low-dose group: 8 APOE mice were fed a high-fat diet and were given a high dose of ultrafine sea cucumber powder by gavage, 1 mg / g, once a day for 8 weeks;

[0107] Samples were collected after 8 weeks of gavage: serum (for 4 lipid tests), heart, liver, and aorta.

[0108] Referring to Cao Xu's experimental method (Cao Xu. Study on extraction process of total triterpenic acids from Ganoderma lucidum spores and exploration of its anti-atherosclerotic effects and mechanisms [D]. Xihua University, 2021.), blood samples should be placed at room temperature for 2 hours and then centrifuged at 3000 rpm for 15 minutes at 4℃. The supernatant can be collected for immediate testing.

[0109] Following the experimental method of Tian Guangjing (Tian Guangjing. Study on the ameliorative effect and mechanism of linseed oil on atherosclerosis [D]. Chinese Academy of Agricultural Sciences, 2018.), aortic tissue was dissected from mice and fixed in 4% paraformaldehyde. The aorta and liver were stained with hematoxylin and eosin (H&E) to assess pathological lesions.

[0110] A high-fat diet can lead to atherosclerosis and cause specific changes in cholesterol. The intervention effects of Example 1 and Comparative Example 1 on high-fat-induced atherosclerosis in mice were evaluated. Serum lipid levels in each group of mice were measured, as shown in Figure 6. Figure 6 shows that the ultrafine sea cucumber powder of Example 1 significantly reduced blood lipid levels in mice. This also confirms that the physical pulverization technology provided by this invention does not alter the bioactivity of sea cucumber polysaccharides.

[0111] Histological changes in the aorta are key alterations in atherosclerosis, primarily manifested as arterial wall thickening and lipid accumulation. Aortic sections (Figure 7) and wall thickness (Figure 8) are shown in Example 1 and Comparative Example 1. It can be seen that mice fed a high-fat diet developed severe aortic tissue lesions, with significant atherosclerotic plaque formation, abundant foam cell formation and accumulation protruding into the lumen, thickening of the aortic intima, and obvious endothelial and medial spaces. The control group showed a significantly increased aortic thickness, reaching 418 μm. Compared to Comparative Example 1, Example 1 demonstrated better intervention efficacy against atherosclerotic disease.

[0112] Comparative Example 3

[0113] Following the preparation method in Example 1, only steps S1, S2, S3, and S4 were performed to obtain the ultrafine sea cucumber powder product.

[0114] Comparative Example 4

[0115] Following the preparation method in Example 1, only steps S1, S2, S3, S4, and S5 were performed to obtain an ultrafine sea cucumber powder product.

[0116] Comparative Example 5

[0117] The preparation method described in Example 1 was followed, except that the supercritical fluid extractor equipped with an improved gas distributor was replaced with a conventional supercritical fluid extractor (without modification to the gas distributor and lacking the design of a second orifice plate). During the extraction process in step S6, pipeline blockage occurred, preventing the extraction from completing properly. This is because the ultrafine sea cucumber powder is too small and easily carried out of the material basket by the carbon dioxide fluid, entering the carbon dioxide pipeline and causing blockage. Furthermore, the incompletely extracted sea cucumber powder still has a strong fishy odor, retains many odorous impurities, and the product purity is not high.

[0118] Following the experimental method of Xu Jialin (Xu Jialin, Peng Jian, Xu Yujuan, et al. Study on physicochemical properties and powder characteristics of mango ultrafine powder with different peel addition amounts [J]. Food and Fermentation Industries, 2024, 1-13.), a 2mL EP tube was filled with sea cucumber powder, and the mass M of the sea cucumber powder in the container was recorded. The bulk density was calculated as follows: Bulk density (g / mL) = M / 2. The larger the bulk density value, the smaller the gap between particles and the smaller the particle size.

[0119] Referring to Li Zhenjiang's experimental method (Li Zhenjiang, Liu Ying, Zhi Li, et al. Effects of ball milling and ultrafine grinding on particle characteristics and polysaccharide extraction of Inonotus obliquus powder [J]. China Food Additives, 2021, 32(11):1-8.), a 2 mL EP tube was filled with sea cucumber powder. The EP tube was continuously vibrated, and sample was added continuously until the powder weight reached a stable state. The mass M1 of the sea cucumber powder in the container was recorded. The tapped density was calculated as follows: Tapped density (g / mL) = M1 / 2. The higher the tapped density, the smaller the interparticle gaps, the smaller the total volume, and the smaller the particle size.

[0120] Following the experimental method of Qi Baokun (Qi Baokun, Liu Yuwen, Yao Yuxue, et al. Analysis of the effects of protease on the volatile flavor of soybean meal based on HS-GC-IMS and HS-SPME-GC-MS [J]. Transactions of the Chinese Society for Agricultural Machinery, 2024, 1-27.), 0.1 g of sea cucumber powder was added to 1 mL of deionized water and placed in a 20 mL headspace vial. After incubation at 60℃ for 30 min, the sample was automatically injected. The injection needle temperature was 85℃, the injection volume was 500 μL, and splitless mode was used.

[0121] Chromatographic conditions: WAX capillary column, column temperature 60℃, operation time 30 min, carrier gas purity N2 ≥ 99.999%, N2 purity ≥ 99.999%. Analytical conditions: 2 mL / min for 2 min, 10 mL / min for 8 min, 100 mL / min for 10 min, 150 mL / min for 10 min, then stop.

[0122] Qualitative analysis of different volatile substances: The drift gas (nitrogen) was set at 150 mL / min. Each spectrum was scanned an average of 12 times. All analyses were performed in triplicate. The retention index (RI) of VC was calculated using N-ketone C4-C9 (Beijing Guoyao Chemical Reagent Co., Ltd.) as an external reference. Qualitative analysis was performed by comparing the RI with the drift time of standards in the GC-IMS library.

[0123] The ultrafine sea cucumber powder prepared in Example 1, and the sea cucumber powder prepared in Comparative Examples 3 and 4, were tested according to the methods described above. The GC-IMS two-dimensional difference topographic maps are shown in Figures 5-7; the darker the color, the higher the concentration of characteristic flavor of the sample. The figures show that the green deodorization technology provided by this invention effectively removes fishy substances from the samples, and the overall flavor difference of the samples after deodorization is not significant. The results of Comparative Examples 3 and 4 show that the ultrafine grinding process increases the concentration of volatile substances in the samples; therefore, further deodorization is needed after ultrafine grinding. However, conventional supercritical fluid extraction cannot complete the deodorization of ultrafine sea cucumber powder.

[0124] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing sea cucumber powder with high polysaccharide release rate, characterized in that, The sea cucumber powder is prepared through the following steps: S1. Sea cucumber pretreatment: Make an opening in the abdomen of the sea cucumber, remove the contents of the sea cucumber body cavity, and wash the sea cucumber body wall with water to obtain pretreated sea cucumber; S2. Enrichment of nutrients in sea cucumbers: The pretreated sea cucumbers obtained in step S1 are temporarily kept in warm water and then dried to obtain dried sea cucumber products. S3. Particle size control of sea cucumber powder: The dried sea cucumber obtained in step S2 is pulverized into coarse sea cucumber powder of 5-30 mesh. S4, "Single" supercritical: The sea cucumber coarse powder obtained in step S3 is placed in the extraction vessel of a supercritical extractor for supercritical carbon dioxide extraction to remove the fishy smell. S5. In-situ ultrafine physical pulverization: The sea cucumber powder that has undergone deodorization in step S4 is ultrafinely pulverized to obtain powder. The bulk density of the powder is controlled to be 0.65-0.70 g / mL, and the tap density is 0.70-0.80 g / mL. S6, "Double" supercritical: The sea cucumber powder obtained in step S5 is placed in the extraction vessel of a supercritical extractor and subjected to supercritical carbon dioxide extraction to remove the fishy smell, resulting in sea cucumber powder with a high polysaccharide release rate. In steps S4 and S6, the parameters of the extraction vessel are set as follows: 30-40℃, extraction pressure: 25-30MPa, CO2 flow rate: 25-30L / h, and extraction time: 2-2.5h. The supercritical extractor mentioned in steps S4 and S6 is a supercritical extractor equipped with a gas distributor; the gas distributor is an improved gas distributor; the improved gas distributor is placed inside the extraction tank. The improved gas diffuser includes an installation pipe (1), a connecting flange (2), a first orifice plate (3), a filter screen (4), a support column (5), a baffle (6), a telescopic rod (7), a spring (70), a pressure plate (8), and a second orifice plate (9). The installation tube (1) is made of transparent material, making it easy to observe the internal condition. Both the upper and lower outer sides of the installation tube (1) are connected to connecting flanges (2) for easy connection to external pipes. The inner middle and lower inner sides of the installation tube (1) are connected to first perforated plates (3). A filter screen (4) is connected to the lower side of each first perforated plate (3). Four support columns (5) are connected between the first perforated plates (3). A baffle (6) is connected to the upper side of each support column (5). The baffles (6) are all connected to the installation tube (1). The first hole plate (3) and the second hole plate (9) are connected to each other. The first hole plate (3) and the second hole plate (9) are the same shape. The second hole plate (9) has twelve conical parts on its upper side to facilitate reflux.

2. The preparation method according to claim 1, characterized in that, The warm water temporary holding process described in step S2 is as follows: the pretreated sea cucumbers are placed in warm water at 50-60℃ for 4-8 hours, and the warm water is changed every 0.5-1 hour.

3. The preparation method according to claim 1, characterized in that, The drying process in step S2 includes freeze drying, heat drying, or air drying.

4. The preparation method according to claim 1, characterized in that, In step S5, pre-cooling is performed before pulverizing using an ultrafine pulverizer; the pre-cooling is performed using dry ice, liquid nitrogen, or by placing the device in a freezing environment for pre-cooling.

5. The preparation method according to claim 1, characterized in that, In step S5, the ultrafine grinding is carried out at a speed of 8000-12000 rpm using a sieve with a mesh size of 0.3-0.6 mm.

6. The preparation method according to claim 1, characterized in that, In step S5, the bulk density is 0.67–0.70 g / mL and the tap density is 0.77–0.80 g / mL.

7. A sea cucumber powder with a high polysaccharide release rate, characterized in that, The sea cucumber powder with high polysaccharide release rate Prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the sea cucumber powder with high polysaccharide release rate as described in claim 7 in the field of food or pharmaceutical preparation.

9. The application according to claim 8, characterized in that, The application in the field of drug preparation is the preparation of drugs for the treatment of atherosclerotic diseases.

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